Collection of the plant sample
The plant sample (Stems and leaves) collection was done in two different seasons- Summer Season and Winter Season from three districts of Assam (Kamrup Metro, Barpeta and Biswanath Chariali, now Sonitpur district). The samples were detached from the plants using sterilized scalpels and were kept in sterilized bags (Sandhu et al., 2014). Since the study did not involve the age of the plant parts hence the samples were collected from neither the tip of the plant nor the older parts of the plant. The Plant samples were then brought to the laboratory the next day for the study.
Ethics Statement
The plant samples were collected from the cultivated areas of the different locations under study and no specific permission was required for the same. The plant materials used in the experiment comply with relevant institutional, national and international guidelines and legislation. Plant samples were collected from the same plants for both the seasons. Only the stems and leaves of the plant samples were collected and hence no uprooting of the plant happened. This study did not involve any endangered or protected species.
Surface Sterilisation
The plant samples were at first washed properly to remove mud and dust particles. For isolating the endophytic species from the plant samples, surface sterilization is required to remove the epiphytes. The samples were first cut into small pieces and then washed in water properly to remove the dust particles. The samples were then treated with distilled water for 1 minute. The samples were then rinsed in 3% Sodium Hypochlorite solution for 30 seconds. The samples were then treated with 70% Ethanol for 60 seconds and thereafter finally rinsed in sterile distilled water (Rani et al.,2017). After this the samples are taken on a clean cheesecloth for soaking.
Isolation and Identification of Endophytic Fungi
Fresh PDA media was made as per the standard protocol (Varghese and Joy,2014) for isolating the endophytic fungi. The media was sterilized in an autoclave at 1210C for 15 minutes. The media was then inoculated with the plant samples for 6 days maintaining the temperature at 28 ± 20C. Streaking fresh culture plates with the isolates was done at regular intervals till the appearance of the pure colonies. The endophytic fungal species were identified morphologically on the basis of colony morphology, shape and size of the spore (Rabha et al., 2015; Nagamani et al.,2016). Some of the Fungal colonies have been represented in Fig. 2.
Isolation and Identification of Endophytic bacteria
Fresh Nutrient Agar Media was made as per the standard protocol (Varghese and joy,2014) for isolating the endophytic bacteria. The media was sterilised and then inoculation with the plant samples was done. The inoculated plates were maintained at a temperature of 25 ± 20C. Streaking was done till the appearance of the pure colonies. For the identification of endophytic bacteria biochemical characterisation followed by Metagenomic sequencing of some of the isolates was carried out.
Biochemical test of Endophytic bacterial isolates
Gram staining, catalase test, Methyl red test, Indole test and citrate utilisation test was performed to study the biochemical nature of the isolates as per the standard protocol (Varghese and Joy, 2014) and has been reported in Table 3. Gram staining was done to differentiate between the Gram positive and Gram negative bacteria (Fig. 1).
Genomic DNA isolation
Genomic DNA of bacterial samples were isolated using a commercially available kit (Invitrogen Pure Link™ Genomic DNA Mini Kit). Isolated genomic DNA was visualised using a 1% agarose gel, stained with ethidium bromide, and visualized under UV light.
Amplification of 16S rDNA
A 1500-bp sequence of bacterial 16S rDNA was amplified with primers 27F and 1492R (Frank, Reich et al. 2008). The 35-cycle amplification was performed in a DNA Thermal Cycler (Eppendorf Master cycler) using 6.25µl of 2X Platinum™ II Hot-Start Green PCR Master Mix (Thermoscientific), 0.25µl of 10uM concentrations of forward and reverse primers, 4µl sterile deionized water, and 2µl DNA template, for a total volume of 12.75µl. The temperature cycling program used was as follows: 1 cycle of pre-denaturation at 94°C for 5 minutes; 35 cycles at 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1.30 minutes; and a final extension of 72°C for 10 minutes. For detection of PCR products, 4µl of the amplified DNA was run on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light.
Sequencing
Impurified PCR product was shipped to Eurofins Genomics (Eurofins Genomics India Pvt. Ltd.) for Sanger sequencing. Purified PCR products were sequenced and analysed on an ABI 3730XL DNA sequencer.
The 16S rRNA gene sequences were compared with other 16S rRNA gene sequences available in GenBank by using the BLASTN program and aligned with similar sequences by using the MUSCLE program embedded in MEGA11. The phylogenetic tree was constructed by applying the neighbour-joining method using the MAGA11 program with 100 bootstraps.
Availability of Data and Material
All Data generated during this study has been mentioned in the paper. The datasets generated during the current study are available at ‘NCBI- GenBank’ with the following Accession numbers to the sequences submitted.
SUB 13361450 Seq1- OQ991266
SUB 13361450 Seq2- OQ991267
The data can be viewed in the weblink of NCBI https:/www.ncbi.nlm.nih.gov
The supporting document related to the metagenomic data for the bacterial identification has been attached as ‘Supplementary Files’.